As the potential for wider varieties of materials to cause cutaneous damage has become recognized, an increasing effort has bene made by industry and environment protection groups to characterize this potential. Federal legislative agencies and commercial manufacturers must consider these risks when developing registering, certifying and shipping materials which could produce toxic effects.
Acute primary irritation is defined as localized reversible inflammatory response of normal living skin to direct injury and is caused by a single application of a chemical agent. Important manifestations are erythema (redness) and edema (swelling). Draize et al., J. Pharmacol. Exp. Ther. 82:337, 1944.
Cumulative irritation is also reversible and consists of primary irritation resulting from repeated exposure of skin to materials that do not cause primary irritation, as reviewed by Guillot et al., Ed. Chem. Toxic 20: 563, 1982.
Corrosion is defined as direct chemical action on normal living skin that results in its disintegration and irreversible alteration on site of chemical contact. According to Draize et al. (ibid.), it is manifested by ulceration or necrosis.
Animals have been used to test for toxic effects. However, the problems inherent in animal testing contribute to variability of all these methods.
1. Intra-laboratory variability in scoring and handling
2. Individual responses vary considerably
3. Application and occlusive seal
4. Sex and age of animals
To evaluate dermal corrosivity, a procedure based on the Department of Transportation Method of Testing Corrosion to skin has been widely used. Code of Federal Regulations, Transportation Title 49, Part 173, Appendix A. Method of Testing Corrosion to the Skin (1983). Six white New Zealand rabbits are shaved, the test substance and a negative control is applied to the shaved skin of each rabbit. Each substances is held in place with a 1.times.1" square 12-ply surgical gauze pad. Corrosion is found if the test sample caused destruction or irreversible alteration of the tissue on at least two of the six rabbits.
In 1977 the United Nations issued special recommendations for class 8 chemicals. (Transportation of Dangerous Goods. Orange Book. Special Recommendations Relating to Class 8, p. 173.) The distinctions between chemicals in Packing Groups I, II and III were given as follows:
Group 1. Substances that cause visible destruction or irreversible alterations of the skin tissue at the site of contact when tested on the intact skin of an animal for a period of not more than three minutes. PA1 Group 2. Substances, other than those in Packing Group 1, that cause visible destruction or irreversible alterations of the skin tissue at the site of contact when tested on the intact skin of an animal for a period of not more than 60 minutes. PA1 Group 3. Substances, other than those meeting Packing Group 1 or 2 criteria, that cause visible destruction or irreversible alterations of the skin tissue at the site of contact when tested on the intact skin of an animal for a period of not more than four hours.
In recent years, different guidelines have evolved in Europe through legislative activity to classify and label potentially dangerous preparations. 1973 Off. J. Eur. Comm. 16 (L189); 1977 Off. J. Eur. Comm. 29 (L303); 1978 Off. J. Eur. Commission 21 (L296). Different levels of skin corrosion define different classifications of the European Commission (EEC, 1983). Sect 4, No. 404 OECD Paris. A substance is corrosive if, when applied to intact animal skin, it produces full-thickness destruction of skin tissue in at least two animals in four hours. If full-thickness destruction occurs within three minutes, the substance is in R35 class, which is comparable to Packing Group I. If full-thickness destruction occurs between three minutes and four hours, the substance is classified as R34, which is comparable to Packing Groups II and III.
In Vitro Alternatives
The state of development of alternative models for dermal irritation and corrosion is improving rapidly. Attempts have been made to utilize other animals but these have not been well received. Attempts to develop true in vitro alternatives have been centered on three approaches. The first alternative uses patches of excised animal skin maintained in a glass diffusion cell (Parish, Fd. Chem. Toxic. 23:278, 1985; and Walker et al., Acta Pharm. Suec. 20:52, 1983). The second approach is to use cultured cells and to measure cytotoxicity. (Lamont et al. In vitro Toxicology: New Directions, Vol. 7, Goldberg (ed.), Mary Ann Liebert, Inc., New York City; Naughton et al. In vitro Toxicology: New Directions, Vol. 7, Goldberg (ed.), Mary Ann Liebert, Inc., New York City).
The third approach uses mathematical SAR models (Free and Wilson, J. Med. Chem. 7:395, 1964; and Goldberg, L., Structure Activity Correlations as a Predictive Tool in Toxicology, Hemisphere Publishing Corp., New York, 1983) or physical parameters for prediction effects (Nago et al., Acta Derm. Venereal Stock 52:11, 1972; and Patrick et al., Tox. and Appl. Pharmacol. 81:476, 1985). One physical parameter frequently described as predictive of corrosivity is pH (Potokar et al., Fd. Chem. Toxicol. 23(6):615, 1985). The analysis of pH and acid/alkali reserve was proposed for classification of preparations as corrosive irritant or not classified as dangerous. High or low pH suggests a test sample will be irritant or corrosive but not how irritating. OECD (1981) recognized that test samples with pH &lt;2 or pH &gt;11.5 are so predictably corrosive that they need not be tested for dermal irritation. However, this basis has incorrectly classified and underestimated corrosive potential and appears to provide only broad guidelines. It only is applicable to test materials which can be called acid or alkali.
All of the foregoing methods for predicting skin corrosion have limitations. The in vitro methods require living cells or isolated tissues or are very limited to specific chemicals such as in SAR. While these procedures do provide an alternative to animal testing, they do not achieve the simplicity and standardization one experiences with other standardized tests. The method of the present invention offers such a test. It provides a standard, quick, reproducible, objective measure of the capacity of materials to cause corrosion.
Scientists have utilized dermal biomacromolecules to study potential effects of chemicals and formulations on the skin for the last forty years. As early as 1953, Scott and Lyon quantified an increase in exposed sulfhydryl groups of keratin after soaps and detergents were applied to the keratin. This exposure resulted from a separation of keratin chains (Van Scott and Lyon, J. Invest. Dermatol. 21:99, 1953). A relationship between the degree of denaturation or separation, the effects of different soaps and detergents on keratin, and the incidence of in vivo dermatitis due to these compounds was observed. Harrold (1959) expanded this work to include investigation of complete formulations on keratin denaturation and separation (J. Invest. Dermatol. 32:581). In 1971, Choman evaluated the swelling response of in vitro skin discs prepared from dermal calf collagen (J. Invest. Derm. 40:177). Sodium lauryl sulfate produced a swelling response, and similar responses for a series of anionic and nonionic surfactants were directly related to their skin irritation potential. Further research clearly established swelling of isolated epidermis and synthetic dermal membranes as a parameter related to irritation. Such swelling is based on adsorption onto and disruption of the three-dimensional keratin protein matrix. Adsorption of the stratum corneum was thoroughly investigated by Imokawa et al., who established a correlation between skin roughness in vivo and in vitro (J. Am. Oil Chem. Soc. 52:475). This and other studies established adsorption as a major step in the initiation of dermal irritation and as perhaps the most important physicochemical parameter in the dermal toxicity of anionic surfactants.
A second major parameter in the initiation of irritation, integrity of the stratum corneum, has been investigated recently. Pemberton and Oliver used monitoring of electrical resistance in skin slices as a measurement of barrier integrity and as an indicator of the corrosive potential of chemicals (Toxic. In Vitro 2:7, 1988). They showed that corrosive agents have a greater ability than noncorrosives to exert a direct physicochemical lytic action on the stratum corneum. Many chemical toxicants produce changes in the keratin barrier matrix in direct proportion to their adsorption to and interaction with the barrier as studies by Van Scott, Harold, Choman and Imokawa (supra). These changes correlate with their potential to produce dermal irritation and corrosion.